Host cell pathways are harnessed and inactivated by viruses during productive infection. In vitro replication systems have been useful for studying viral DNA replication mechanisms, but they fail to capture the full complexity of the cellular environment and the importance of spatial regulation and intracellular host defenses. It is clear from recent work that the cellular DNA damage machinery impacts virus infection. Although some cellular proteins involved in regulating virus-host interactions have been identified, a comprehensive analysis of host replication and repair factors has not yet been undertaken. The advent of RNA interference (RNAi) and the development of short interfering RNA (siRNA) libraries, enables a systematic analysis of host genes involved in viral processes. In this application we propose to use a targeted siRNA library against cellular proteins involved in DNA replication, repair and recombination. We will compare and contrast four well- characterized DNA viruses that replicate in the nucleus. These representatives of different viral families and genomes are: the linear double-stranded DNA (dsDNA) genomes of Adenovirus (Ad) and Herpes Simplex Virus type 1 (HSV-1), the single-stranded DNA (ssDNA) genome of the parvovirus Adeno-Associated Virus (AAV), and the circular dsDNA genome of Simian Virus type 40 (SV40). We will develop and validate high- throughput functional assays for transduction, replication, and virus production for the four viral systems, using either wild-type viruses or recombinant vectors that express reporter proteins. Hits that emerge from our screens will be confirmed by additional siRNAs to rule out off-target effects, together with verification of knock- down at the RNA and protein level. These screens will yield an important dataset of information about host factors that regulate virus infection and will provide a platform for further studies into the mechanisms that control virus infection. Comparing multiple viral systems in parallel will reveal both unifying approaches and alternative strategies employed by viruses with different genomes, and will identify host factors that both positively and negatively regulate virus infection. Understanding these themes will provide insights into cellular DNA repair pathways that ensure the integrity of the host genome, and may identify new targets for antiviral approaches. PUBLIC HEALTH RELEVANCE: Viruses are dependent upon the host cell machinery for replication of their genetic material and progeny production, and therefore present powerful tools to study cellular processes. We propose to screen four different DNA viruses (adenovirus, herpesvirus, adeno-associated virus and simian virus 40) against a library of small interfering RNAs targeted to cellular proteins involved in DNA replication, recombination and repair. These screens will identify cellular factors that regulate viral infection, will provide insights into virus-host interactions and fundamental cellular processes, and may suggest targets for anti-viral therapies.